Bi-or multifunctional molecules based on a dendroaspin scaffold

ABSTRACT

Dendroaspin, a polypeptide neurotoxin analogue is modified by recombinant DNA techniques, particularly “loop grafting” to provide a modified polypeptide. The modified polypeptide is constructed so as to retain dendroaspin activity, for example, platelet adhesion to fibrinogen, in addition to possessing one or more further biological or biochemical activities not native to dendroaspin, for example, platelet derived growth factor (PDGF) activity or hirudin activity.

This application is a national stage application of PCT/GB 98/00848,filed Mar. 20, 1998, which claims foreign priority to GB 97 05787.1filed Mar. 20, 1997.

The present invention relates to dendroaspin-based chimeric moleculeswhich have anticoagulant, antiplatelet and other activities. Theinvention also relates to nucleic acid molecules encoding these chimericdendroaspin molecules, cloning and expression vectors comprising suchnucleic acids and host cells transformed with expression vectors so asto provide recombinant chimeric multifunctional dendroaspin. Theinvention further relates to pharmaceutical compositions comprisingchimeric dendroaspin for use in the prevention or treatment of diseaseassociated with thrombus formation or platelet aggregation. Theinvention also further relates to the use of a dendroaspin scaffold inthe design and generation of chimeric dendroaspin derivatives havinginhibitory activity against integrin binding activity of platelets plussome further functionality such as an anticoagulant or antithromboticaction.

The role of blood coagulation is to provide an insoluble fibrin matrixfor consolidation and stabilization of a haemostatic plug. Formation ofa cross-linked fibrin clot results from a series of biochemicalinteractions involving a range of plasma proteins.

Acute vascular diseases, such as myocardial infarction, stroke,pulmonary embolism, deep vein thrombosis and peripheral arterialocclusion are caused by either partial or total occlusion of a bloodvessel by a blood clot.

The formation of a blood clot within a blood vessel is termed thrombosisand is dependent upon platelet aggregation. In the context of bloodvessel injury (such as that which might arise in surgical procedures)the interaction of blood platelets with the endothelial surface ofinjured blood vessels and with other platelets is a major factor in thecourse of development of clots or thrombi.

Various agents for preventing formation of blood clots are nowavailable, such as aspirin, dipyridamole and filopidine. These productsgenerally inhibit platelet activation and aggregation, or delay theprocess of blood coagulation but they have the potential side effect ofcausing prolonged bleeding. Moreover, the effect of such products canonly be reversed by new platelets being formed or provided.

Platelet aggregation is dependent upon the binding of fibrinogen andother serum proteins to the glycoprotein receptor IIb/IIIa complex onthe platelet plasma membrane. GP IIb/IIIa is a member of a large familyof cell adhesion receptors known as integrins, many of which are knownto recognize an Arg-Gly-Asp (RGD) tripeptide recognition sequence.

Heparin and low molecular weight heparins have been used widely to treatconditions, such as venous thromboembolism, in which thrombin activityis responsible for the development or expansion of a thrombus. Althougheffective, heparin produces many undesirable side effects, includinghaemorrhaging and thrombocytopenia. A more specific and less toxicanticoagulant is therefore required.

Direct thrombin inhibitors are available and examples of these arehirudin, hirugen and hirulog (the latter two being synthetic hirudinderivatives), PPACK (a synthetic tripeptide) and argatroban (an argininederivative). The actions of these inhibitors are reviewed in Lefkovits Jand Topol E J (1994), Circulation 90:1522-1536. Although in theory, thebleeding risk with direct thrombin inhibitors is lower than with otherantithrombotics because of their mono-target specificity, absence ofdirect platelet effects, and short half-life, bleeding still remains asthe most concerning adverse effect.

There are a range of other thrombin inhibitors which have been developed(listed in table 1 of Lefkovits J and Topol E J supra) but these haveturned out to be just too toxic for clinical use.

Localized narrowing of an artery caused by atherosclerosis is acondition which can usually be remedied surgically by the technique ofballoon angioplasty. The procedure is invasive and causes some tissuedamage to the arterial wall which can result in thrombus formation.Extracellular proteins such as fibronectin in the arterial wall becomeexposed to blood in the artery. Platelets bind to the RGD motif offibronectin via integrin receptors which in turn leads to plateletaggregation and the start of the cascade of clotting reactions. An agentwhich specifically inhibits platelet aggregation at the sites of damageand which also inhibits clotting at these sites is required. The agentshould be non-toxic and free of undesirable side effects such as a riskof generalized bleeding.

Integrins are a family of cell surface receptors that mediate adhesionof cells to each other or to the extracellular matrix (Kieffer N &Philips D R (1990) Annu Rev Cell Biol 6: 329-357; Hynes R O (1992) Cell69: 11-25; McEver R P (1992) Curr Opin Cell Biol 4: 840-849; Smyth S Set al (1993) Blood 81: 2827-2843; Giancotti F G and Mainiero F (1994)Biochim Biophys Acta 1198: 47-64). They are composed of noncovalentlyassociated α and β transmembrane subunits. There exist 16 different αand β different β subunits that heterodimerize to produce about 20different kinds of receptors (Clark E A & Brugge J S (1995) Science 268:233-239). Among the integrins, the platelet membrane integrin α_(IIb)β₃is one of the best characterized. Upon cell activation, the α_(IIb)β₃integrin binds several glycoproteins, predominantly through theArg-Gly-Asp (RGD) tripeptide sequence (Pierschbacher M D and Ruoslahti E(1984) supra; Plow E F et al (1987) Blood 70: 110-115; Pytela R et al(1986) Science 231: 1559-1562) present in fibrinogen (Nachman R L andNachman L L K (1992) J Clin Invest 69: 263-269), fibronectin (Gardner JM and Hynes R O (1985) Cell 42: 439-448), von Willebrand factor (RuggeriZ et al (1983) J Clin Invest 72: 1-12), vitronectin (Pytela R M et al(1985) Proc Natl Acad Sci USA 82: 5766-5770), and thrombospondin(Karczewski J et al (1989) J Biol Chem 264: 21322-21326). The nature ofthe interactions between these glycoprotein ligands and their integrinreceptors is known to be complex, and conformational changes occur inboth the receptor (Sims P J et al (1991) J Biol Chem 266: 7345-7352) andthe ligand (Ugarova T et al (1995) Thromb Haemostasis 74: 253-257).

Recently, many proteins from a variety of snake venoms have beenidentified as potent inhibitors of platelet aggregation andintegrin-dependent cell adhesion. The majority of these proteins whichbelong to the so-called “disintegrin” family share a high level ofsequence homology, are small (4-8 kDa), cysteine-rich and contain thesequence RGD (Gould R J et al (1990) Proc Soc Exp Biol Med 195: 168-171)or KGD (Scarborough R M et al (1991) J Biol Chem 266: 9359-9362). Inaddition to the disintegrin family, a number of non-disintegrin RGDproteins of similar inhibitory potency, high degree of disulfidebonding, and small size have been isolated from both the venoms of theElapidae family of snakes (McDowell R S et al (1992) Biochemistry 31:4766-4772; Williams J A et al (1992) Biochem Soc Trans 21: 73S) and fromleech homogenates (Knapp A et al (1992) J Biol Chem 267: 24230-24234).All of these proteins are approximately 1000 times more potentinhibitors of the interactions of glycoprotein ligands with the integrinreceptors than simple linear RGD peptides; a feature that is attributedto an optimally favourable conformation of the RGD motif held within theprotein scaffold. The NMR structures of several inhibitors includingkistrin (Adler M et al (1991) Science 253: 445-448; Adler M and Wagner G(1992) Biochemistry 31: 1031-1039; Adler M et al (1993) Biochemistry 32:282-289), flavoridin (Senn H and Klaus W (1993) J Mol Biol 234:907-925), echistatin (Saudek V et al (1991) Biochemistry 30: 7369-7372;Saudek V et al (1991) Eur J Biochem 202: 329-328; Cooke R M et al (1991)Eur J Biochem 202: 323-328; Cooke R M et al (1992) Protein Eng 5:473-477), albolabrin (Jaseja M et al (1993) Eur J Biochem 218: 853-860),decorsin (Krezel A M et al (1994) Science 264: 1944-1947), anddendroaspin (Jaseja M et al (1994) Eur J Biochem 226: 861-868; SutcliffeM J et al (1994) Nature Struct Biol 1: 802-807) have been reported, andthe only common structural feature elucidated so far is the positioningof the RGD motif at the end of a solvent exposed loop, a characteristicof prime importance to their inhibitory action.

Recent studies have implied a role for the amino acids around thetripeptide RGD in regulating the ligand binding specificity shown bysnake venom proteins. Scarborough R M et al (1993) J Biol Chem 268:1058-1065 examined a range of disintegrins and observed that thosecontaining RGDW were very effective at inhibiting the interactions offibrinogen to purified α_(IIb)β₃ but not of vitronectin and fibronectinto purified α_(v)β₃ and α₅β₁, respectively, whereas the converse wastrue for disintegrins containing the sequence RGDNP. Other regions ofamino acid sequence divergence may also be contributory (Scarborough etal (1993) supra).

Dendroaspin, a short chain neurotoxin analogue containing the RGDsequence, and the disintegrin kistrin, which show little overallsequence homology but have similar amino acids flanking the RGD sequence(PRGDMP), are both potent inhibitors of platelet adhesion to fibrinogenbut poor antagonists of the binding of platelets to immobilizedfibronectin (Lu X et al (1994) Biochem J 304: 929-936). In contrast,elegantin, which has 65% sequence homology to kistrin but markedlydifferent amino acids around RGD (ARGDNP), preferentially inhibitedplatelet adhesion to fibronectin as opposed to fibrinogen and binds toan allosterically distinct site on the α_(IIb)β₃ complex.

Smith J W et al (1995) Journal of Biological chemistry 270: 30486-30490undertook protein “loop grafting” experiments to construct a variant oftissue-type plasminogen activator (t-PA) which bound platelet integrinα_(IIb)β₃. Amino acids in a surface loop of the epidermal growth factor(EGF) domain of t-PA were replaced with residues from acomplementarity-determining region (CDR) forming one CDR of a monoclonalantibody reactive against the adhesive integrin receptor α_(IIb)β₃. Theresulting variant of t-PA (loop-grafted-t-PA) bound α_(IIb)β₃ withnanomolar affinity and had full activity to both synthetic and naturalsubstrates. The effects and applicability of loop grafting arealtogether unpredictable and uncertain.

The present inventors have now discovered that the dendroaspin scaffoldlends itself to modification. When dendroaspin (including the RGD motif)is modified to incorporate further functional amino acid sequences forexample active portions or motifs of agonists, antagonists or inhibitorsof factors in the clotting cascade, the resulting molecules areparticularly useful as anticoagulants and do not suffer from thedrawbacks associated with existing anticoagulants.

In first aspect the present invention provides a hybrid polypeptidecomprising a first amino acid sequence including the RGD motif andconferring dendroaspin activity and a further amino acid sequenceconferring activity other than that of dendroaspin activity.

The invention also provides a hybrid polypeptide having integrin bindingactivity comprising a dendroaspin scaffold and a further non-dendroaspinamino acid sequence, preferably of different activity.

Advantageously, the molecules of the invention have an integrin bindingactivity which when administered in vivo results in the binding of themolecules to platelets thereby inhibiting the aggregation of theplatelets, at sites of injury. Moreover, the non-wild-type dendroaspindomain provides secondary, optionally further functionality egantithrombotic action, inhibiting cell migration and proliferation andregulating signal transduction. Molecules of the invention are thereforebi- or multifunctional in their activities against blood coagulation,particularly thrombus formation and arterial/venous wall thickening atthe sites of injury. Polypeptides of the invention may have activitiesagainst leukocyte recruitment, immune system activation, tissue fibrosisand tumorigenesis.

The polypeptide of the invention may comprise at least two said furtheramino acid sequences, preferably the two said further sequences are thesame.

The further amino acid sequence may comprise two or more amino acidsequence portions separated by at least one amino acid residue ofdendroaspin. The two or more sequence portions may be transposed withrespect to one another and to the linear order of amino acids in thenative further amino acid sequence. In other words, the native order ofthe two or more amino acid sequence portions is altered although theactual sequence of each portion may not necessarily be altered.

The said further sequence may be selected from platelet derived growthfactor (PDGF), glycoprotein (GP) IBα, hirudin, thrombin, throinbomodulin(particularly the fifth EGF-like domain thereof), vascular epidermalgrowth factor (VEGF), transforming growth factor-β1 (TGFβ1), basicfibroblast growth factor (bFGF), angiotensin II (Ang II), factor VIIIand von willebrand factor (vWF).

In this way the molecules of the invention may be renderedmultifunctional so that they are active against more than just plateletaggregation, for example another component in the clotting cascade (egthrombin activity), or the intracellular signaling cascade (eg growthfactor). The modified dendroaspins of the invention may he engineered sothat the further amino acid sequence has integrin binding activity,hereby providing a dendroaspin based molecule with augmented integrinbinding activity.

The polypeptide of the invention preferably comprises an amino acidsequence as shown in FIG. 3 (SEQ ID NO:4). Prior to inclusion of saidfurther amino acid sequence the dendroaspin scaffold of the inventionincludes homologous molecules which may share about 50% amino acidsequence homology, preferably about 65%, more preferably about 75% andeven more preferably about 85% homology with dendroaspin.

Excluding the nucleic acid sequence encoding said further amino acidsequence nucleic acid sequences encoding the polypeptide of theinvention may share about 50% nucleotide sequence homology, preferablyabout 65%, more preferably about 75% and even more preferably about 85%homology with a dendroaspin nucleotide sequence.

The polypeptides of the invention may comprise a greater or lessernumber of amino acid residues compared to the 59 amino acids ofdendroaspin. For example, the molecules of the invention may comprise anumber of amino acid residues in the range 45 to 159, preferably about49 to 85, more preferably about 53 to 69, even more preferably about 57to 61.

The further amino acid sequence is preferably incorporated into (a) loopI and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loopIII; or (d) loop I, loop I and loop III of the dendrcaspin scaffold.Loop I comprises amino acid residues 4-16, loop II residues. 23-36 andloop III residues 40-50. However, the further amino acids beingincorporated may extend into or substitute regions external to theloops, ie residues 1-3, 17-22 and 37-39 such that residues of thenon-loop regions are augmented or substituted for those of the furtheramino acid sequence or sequences being inserted.

The further amino acid residues are preferably incorporated into eitherloop I or loop II. In this way the RGD-containing loop III is unalteredand so the integrin binding function of dendroaspin is retained.

A further RGD motif may be introduced into the dendroaspin scaffold,preferably into loop I or loop II, thereby increasing dendroaspinactivity.

A preferred location for the inserted further sequence is at a site indendroaspin scaffold between amino acid residues: 4-16, 18-21, 23-36, or52-59.

Each inserted further amino acid sequence or portion of a further aminoacid sequence is preferably an amino acid sequence in the range 3-40amino acid residues, more preferably 3-16, even more preferably 3-14amino acid residues long. The start of the inserted further amino acidsequence may be at any one of amino acid residues 1-57 of thedendroaspin scaffold. The end of the inserted further amino acidsequence may be at any one of amino acid residues 3-59 of thedendroaspin scaffold.

When two further amino acid sequences are inserted into the dendroaspinscaffold then the linear distance between these is preferably in therange 1-35 amino acids, more preferably 1-14 amino acids. When more thantwo further amino acid sequences are inserted then there is preferablyat least one native dendroaspin amino acid residue separating eachfurther amino acid sequence.

The RGD-containing loop may be modified by insertion, deletion orsubstitution of one of more amino acid residues, preferably a maximum of8 or a minimum of 1 amino acids can be modified within loop III ofdendroaspin.

The RGD loop preferably has an amino acid sequence as shown in FIG. 3B(residues 40-50 of SEQ ID NO:4). An advantage of modifying the RGD loopregion is that the integrin binding activity may be enhanced and becomemore specific for certain glycoprotein ligands. Also, if one or more ofthe “foreign” further amino acid sequences grafted into the dendroaspinscaffold has steric effects on the RGD motif then loop III around theRGD site can be modified to overcome any steric hindrance therebyrestoring, perhaps enhancing RGD functionality.

Loop I and/or loop II may be modified by insertion, deletion orsubstitution of one or more amino acid residues. Any suitable number ofamino acids can be inserted into the dendroaspin scaffold to give thedesired bi- or multi-functional activity although a number of residuesin the range 14 to 36 are preferred for insertion at one or more sitesin the dendrcaspin scaffold.

Modification of the loops may become necessary if a “foreign” furtheramino acid sequence grafted into the dendroaspin scaffold has a sterichindrance effect either on another grafted domain or on theRGD-containing loop. Computer assisted molecular modelling using InsightII software (Molecular Simulations Inc) can be used to predict thestructure of the “loop grafted” dendroaspins of this invention. Ininstances where steric effects between the loops may serve to cause lossof functionality, these effects can be “designed out” by modifyingappropriate parts of the dendroaspin molecule in an appropriate way.Sometimes this may involve inserting a number of suitable amino acidresidues to extend one or more of the loop structures.

Preferred modification includes the insertion of polyglycine into theloop or loops of the dendroaspin scaffold in order to extend them. Othermodifications comprising repeat units of an amino acid residue or numberof residues can be used. Computer modelling studies can be used todesign the loop modifications needed in order to extend the loops ofdendroaspin.

In the design of a bifunctional or multifunctional molecule inaccordance with the invention, “fine tuning” of activity, stability orother desired biological or biochemical characteristic may be achievedby altering individual selected amino acid residues by way ofsubstitution or deletion. Modification by an insertion of an amino acidresidue or residues at a selected location is also within the scope ofthis “fine tuning” aspect of the invention. The site-directedmutagenesis techniques available for altering amino acid sequence at aparticular site in the molecule will be well known to a person skilledin the art.

In second aspect the invention provides a nucleic acid molecule encodinga polypeptide as hereinbefore defined.

The nucleic acid may be linked operatively to a promoter and optionallyto a nucleic acid sequence encoding a heterologous protein or peptidethereby to encode a fusion product. The promoter is preferablyβ-D-isopropyl-thiogalactopyranoside (IPTG) inducible and theheterologous protein or peptide may be glutathione S-transferase (GST).

This aspect of the invention also includes a plasmid comprising anucleic acid as hereinbefore defined. The plasmid is preferably pGEX-3X.

In third aspect the invention provides a host cell transformed with aplasmid as hereinbefore defined, preferably said host cell is E coli.

The invention therefore also provides a cell culture comprisingtransformed host cells as hereinbefore defined.

In fourth aspect the invention provides a method of producing apolypeptide as hereinbefore defined comprising culturing a host cell ashereinbefore defined so as to express said polypeptide, extracting thepolypeptide from the culture and purifying the same.

In fifth aspect, the invention provides a method of producing amultifunctional anticoagulant comprising the steps of:

a) constructing an expression vector comprising a nucleic acid sequenceencoding a dendroaspin scaffold operatively linked to a promoter andoptionally linked to nucleic acid encoding an heterologous protein forco-expression therewith.

b) modifying at least a portion of the nucleic acid sequence of thevector encoding the dendroaspin scaffold, excluding the RGD motif, byone or more of insertion, deletion or substitution of nucleic acidresidues so that on expression the dendroaspin scaffold comprises afurther amino acid sequence of activity other than that of dendroaspinactivity.

c) transforming a host cell with the vector and causing the host cell toexpress the modified dendroaspin nucleic acid sequence.

The method preferably further comprises the steps of:

d) extracting the modified dendroaspin from a host cell culture,

e) purifying the modified dendroaspin from the cell culture extract,optionally including the step of cleaving the dendroaspin from aco-expressed heterologous protein.

The heterologous protein is preferably GST and the purificationpreferably involves affinity chromatography using glutathione Sepharose4B contained in the GST purification modules followed by factorXa-mediated cleavage of the modified dendroaspins from GST.

The invention therefore provides a polypeptide as hereinbefore definedobtainable by the method of producing a multifunctional anticoagulant asdefined above.

In sixth aspect the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of a polypeptide ashereinbefore defined, optionally further comprising a pharmaceuticallyacceptable excipient or carrier. A multiplicity of polypeptides of theinvention of different functionalities may be combined together in apharmaceutically acceptable form so as to provide a desired treatment. Amultiplicity of polypeptides of the invention of differentfunctionalities may be combined together in a pharmaceuticallyacceptable form so as to provide a desired treatment.

The polypeptide of the invention is preferably formulated forintravenous injection or intravenous infusion although other methods ofadministration are possible, eg subcutaneous or intramuscular should itbe desired to provide a slow release into the circulatory system of anindividual. Also possible is the formulation of the polypeptide for usewith implanted controlled release devices such as those used toadminister growth hormone for example.

One formulation may comprise extravasated blood combined with apolypeptide of the invention at a concentration in the range 1 nM-60 μM.This blood may be stored in ready to use form and provides an immediateand convenient supply of blood for transfusion in cases when clottingmust be avoided such as during or immediately following surgicalprocedures.

In seventh aspect the invention provides a polypeptide as hereinbeforedefined for use as a pharmaceutical.

In eighth aspect the invention provides for the use of a polypeptide ashereinbefore defined for the manufacture of a medicament for thetreatment of disease associated with thrombosis; more particularlythrombosis, myocardial infarction, retinal neovascularization,endothelial injury, dysregulated apoptosis, abnormal cell migration,leukocyte recruitment, immune system activation, tissue fibrosis andtumorigenesis.

The invention also provides methods for the treatment of diseaseassociated with thrombosis; more particularly thrombosis, myocardialinfarction, retinal neovascularization, endothelial injury, dysregulatedapoptosis, abnormal cell migration, leukocyte recruitment, immune systemactivation, tissue fibrosis and tumorigenesis. The methods compriseadministering a therapeutically effective amount of a polypeptide ashereinbefore defined.

Preferred embodiments of the invention will now be described and withreference to the following examples and drawings in which:

FIG. 1 shows the basic three-dimensional structure of a molecule ofdendroaspin. N denotes the —NH terminus and —C denotes the —COOHterminus. Amino acid residues are numbered.

FIG. 2A shows the nucleotide (SEQ ID NO:2) and corresponding amino acidsequences (SEQ ID NO:1) of dendroaspin. Individual syntheticoligonucleotides are indicated by numbers 1-10.

FIG. 2B is a partial restriction map of plasmid pGEX-DEG.

FIG. 3A (SEQ ID NOS:4-22) comprises alignments of modified dendroaspinswhere the inserted amino acid sequences are listed beneath the aminoacid sequence of dendroaspin.

FIG. 3B (SEQ ID NOS:4, 23-42) is like FIG. 3A and further alignments ofthe amino acid sequences (one letter code) of various modifieddendroaspins.

FIG. 3C (SEQ ID NOS:4, 43-46, 7) is like FIG. 3A and shows modifiedmolecules of the invention.

FIG. 3D is a table showing various activities of the modified moleculesof the invention.

FIG. 4 shows the expression plasmid pGEX-3X (SEQ ID NOS:47-48) used toclone and express modified dendroaspin fusion protein.

FIG. 5A shows a 12.5% SDS-PAGE gel comparing the total cellular lysatesof either induced (lane 1) or uninduced (lane 2) bacteria transformedwith the recombinant plasmid pGEX-DEG harbouring the modifieddendroaspin gene.

FIG. 5B shows a 10% SDS-PAGE gel comparing total cell lysate (lane 1)from induced bacteria transformed with pGEX-DEG and an affinity purifiedextract of such a lysate (lane 2).

FIG. 5C shows a 20% SDS-PAGE gel of affinity purified and factor Xadigested GST-modified dendroaspin fusion protein. Lane 1 is dendroaspin,lane 2 is GST-Den and lane 3 is factor Xa digested GST-Den

FIG. 6A is a reverse phase HPLC elution profile of recombinantdendroaspin purified on a Vydac C₁₈ column developed with anacetonitrile gradient (the retention time of recombinant dendroaspin at44 minutes was indicated by an arrow).

FIG. 6B is a reverse phase HPLC elution profile of the peak fractioneluting at 44 minutes in FIG. 6A carried out under similar conditions.

FIG. 7 shows immunoprecipitation of dendroaspin containing PDGF loop Iusing antibodies R38 and R65 raised to loop I and R51 raised to bothloop I and loop III. A 32 kDa protein which corresponds to theGST-mutant dendroaspin was observed. Lane 1 is DEN-PDGF probed with R38,lane 2 probed with R65 and lane 3 probed with R51.

FIG. 8 is a bar chart showing the results of an experiment in whichPDGF-dendroaspin is used to inhibit PDGF induced proliferation of humanfibroblast cells. “Peptide 1” corresponds to the linear amino acidsequence of PDGF loop I.

Dendroaspin is a short chain neurotoxin homologue from the venom ofElapidae snakes, which lacks neurotoxicity. Unlike neurotoxins, itcontains an Arg-Gly-Asp-(RGD)-motif and functions as an inhibitor ofplatelet aggregation and platelet adhesion with comparable potency tothe disintegrins from the venoms of Viperidae. The structure ofdendroaspin in solution has been determined using NMR spectroscopy(Sutcliffe M J et al (1994) Nature Struct Biol 1: 802-807). Thestructure contains a core similar to that of short chain neurotoxins,but with a novel arrangement of loops and a solvent-exposed RGD-motif.Dendroaspin is thus an integrin antagonist with a well defined folddifferent from that of the disintegrins, based on the neurotoxinscaffold.

The structure of dendroaspin consists of a core region from which threeloops, denoted I, II and III (residues 4-16, 23-36 and 40-50 of SEQ IDNO:4) extend outwards (FIG. 1). The core contains the four disulphidebonds which are spatially close to each other and hold the loopstogether. The amino acid sequence of dendroaspin (SEQ ID NO:1) is shownin FIG. 2A.

In the following examples materials used include:

Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, IPTG(isopropyl-β-D-thio-galactopyranoside) and DH5α competent cellspurchased from Life Technologies Ltd (UK) or Promega Ltd (Southampton,UK). Vent (exo-) DNA polymerase was supplied by New England Biolabs Ltd(Hitchin, UK). Oligonucleotides were made either in King's CollegeSchool of Medicine & Dentistry (London, UK) or by Cruachem Ltd (Glasgow,UK) and further purified by denaturing PAGE on a 15% acrylamide/8 M ureagel. Deoxynucleotide triphosphates (dNPT's), dideoxynucleotidetriphosphates (ddNTP's) and plasmid pGEX-3X, a vector that expresses acloned gene as a fusion protein linked to glutathione S-transferase(GST) and Glutathione-Sepharose CL-4B were purchased from PharmaciaBiotech Ltd (Herts, UK). “Geneclean” kit and Plasmid maxi kit werepurchased from Bio 101 (La Jolla Calif., USA) and Qiagen Ltd (Surrey,UK) respectively. The sequencing Sequenase 2.0 was obtained fromCambridge Bioscience (Cambridge, UK). [³⁵S]dATP[αS] and ¹²⁵I (15.3mCi/mg iodine) were supplied by NEN Dupont (Herts, UK) and AmershamInternational Plc (Amersham, Bucks, England) respectively.

EXAMPLE 1 Construction of Expression Vectors Encoding Variants ofDendroaspin

The wild type dendroaspin gene was inserted into a plasmid pGEX-3X (FIG.2) successfully and expressed according to the method of Lu et al,(1996) J Biol Chem 271: 289-295. Starting with the wild type gene fordendroaspin, variants of dendroaspin genes were engineered usingrecombinant DNA technology. For the longer insertion variants,oligo-nucleotides which encode the non-dendroaspin or heterologous aminoacids were simply inserted directly into suitably restriction digestedwild type dendroaspin gene and then ligated. For minor changes such asmodification of a few amino acid residues including the insertion,substitution or deletion, the Transformer™ Site-Directed Mutagenesis kitfrom Clonetech Laboratories was used in accordance with themanufacturers instructions.

FIG. 2A shows the nucleotide sequence of the synthetic dendroaspin (Den)gene (SEQ ID NO:2). The gene was designed on the basis of the knownamino acid sequence (Williams J A et al ((1992)) Biochem Soc Trans 21:73S) and the codons for each amino acid were adopted from those whichwere highly expressed in E coli (Fiers W ((1982)) Gene 18: 199-209) Tensynthetic oligonucleotides are shown in brackets and numberedindividually 1 to 10 either above the coding strand or below thenon-coding strand. The stop codon is indicated by an asterisk.Three-letter amino acid code is used and the total of 59 amino acids ofDen are only numbered 1 for N-terminal residue arginine and 59 forC-terminal leucine.

FIG. 2B is a partial restriction map of pGEX-DEG. Only the dendroaspin(Den)gene and its relevant upstream region are shown.

FIG. 3A shows the amino acid sequences of various modified dendroaspinmolecules (SEQ ID NOS:5-22) which will be described in more detailbelow. In each case the modified inserted residues are set out beneaththe amino acid sequence of dendroaspin (SEQ ID NO:4).

EXAMPLE 2 Modified Dendroaspin Containing a Platelet-derived GrowthFactor (PDGF) Domain

Platelet-derived growth factor (PDGF) is a 30-kDa polypeptide and amajor serum mitogen and chemotactic factor for mesenchymal cells.Originally purified from human platelets, PDGF has subsequently beenfound to be produced by other cell types such as smooth muscle cells,placental cytotrophoblast cells, fibroblasts and connective tissue. PDGFhas been shown to promote cell migration and proliferation and these arekey events in natural processes such as embryogenesis and wound healing.PDGF has also been implicated in a number of pathological states, suchas arteriosclerosis, fibrosis and rheumatoid arthritis (Heldin C H andWestermark B (1990) Cell Regul 1: 555-566 and Engstrom U et al (1991) JBiol Chem 267: 16581-16587). PDGF is a dimer of two similar chainscalled A- and B-chains.

Amino acid sequences derived from PDGF B chain is inserted into loop IIof dendroaspin as shown in FIGS. 3A (SEQ ID NOS:6-10) and 3C (SEQ IDNO:7), maintaining the adhesive property of the RGD sequence of loop IIIwithin dendroaspin. The modified dendroaspins are then examined forcompetitive inhibition of PDGF activity by analyzing smooth muscle andfibroblast cell proliferation and integrin antagonist activity. FIG. 3Dsummarizes the results obtained with the example given in FIG. 3C. Themodified dendroaspin molecules inhibit ADP-induced platelet aggregationand PDGF-induced proliferation of fibroblast cells.

Assembly and cloning of the gene of dendroaspin containing PDGF loop I:The gene of dendroaspin containing PDGF loop I was assembled from thefragments (77 mer, 76 mer, 42 mer and 44 mer) of wild-type gene, afterdigestion with Bam HI, EcoR I, Hinf I and Hpa II, and a pair ofcomplementary phosphorylated mutagenesis oligos (81 mer and 80 mer). Amixture of the total 6 fragments was heated to 85° C. for 5 min andannealed by slowly cooling down to room temperature (RT). Ligation wasperformed at 16° C. for 15 hours in a total volume of 50 μl containingabout 1 mM of each fragment, 50 mM Tris-HCl (pH7.6), 10 mM MgCl₂, 1 mMDTT, 1 mM ATP and 5% PEG 8000 and 5 units of T4 ligase. After ligation,1 μl of ligation mixture was used as template together with two5′-overhanging oligonucleotides as primers and 2 units of Ventpolymerase for PCR. The single product obtained was of expected sizechecked by 2% agarose gel. The hybridisation gene was then cloned intothe vector pGEX-3X to produce the recombinant plasmid pGEX-3X comprisingPDGF-niodified dendroaspin gene.

Transformation and plasmid DNA isolation: The pGEX-3X vector comprisingthe PDGF-modified dendroaspin gene (about 5 ng) was used to transform 50μl of the E coli strain DH5α. controls were set up of a vial containingcompetent cells only, a vial containing unligated plasmid DNA and a vialcontaining wild-type plasmid DNA. The incubation media used for thebacteria is SOC medium (containing 20 g/l bacto-tryptone, 5 g/lbacto-yeast extract, 0.5 gl NaCl, 2.5 mM KC, 20 μM MgCl₂ and 20 mMglucose). The transformation step was followed by a recovery period inwhich the bacterial culture was incubated without ampicillin for anhour. After transformation, a sufficient quantity of each of thebacterial cultures was plated out. SOC agar medium containing ampicillinwas used to culture the bacteria. The ampicillin selection marker ofpGEX-3X confers ampicillin resistance on any transformed bacteriaallowing them to grow whilst any untransformed bacteria are eliminated.The control plate remained clear following incubation overnight.

Individual colonies were picked from the plates, each one representing amodified dendroaspin gene. These were cultured on SOC media containingampicillin for a few hours. A small amount of each was removed andtransferred to new SOC media containing ampicillin. Sterile glycerol wasadded to the original bacterial culture to permit storage at −70° C. asa stock supply. The remaining cultures we re incubated overnight. Theisolation of the plasmid DNA from the transformed bacterial culture iscarried out by a plasmid mini prep (for quick test) or maxi-prep (forDNA sequencing) following the manufacturer's instructions (QIAGEN). Aplasmid mini-prep was carried out on each to isolate the plasmid DNAonce more; the majority of which comprised the modified dendroaspingene. DNA sequencing was carried out on the region of the plasmidcontaining the dendroaspin gene fragment to check that the modificationwas present and correct. Complete DNA sequencing of the insertedfragments was made using the dideoxy chain-termination method of Sangeret al (1977) Proc Natl Acad Sci 74: 5463-5467.

Protein expression: E coli DH5α was transformed with modified plasmidpGEX-3X using standard methods particularly as described in Sambrook etal (1989) Molecular cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, N.Y. The transformed cultures were used for proteinexpression. Medium were inoculated with an overnight seed culture (1%,v/v) and grown in LB/ampicillin medium (100 μg/ml) and shaken at 37° C.until an A₆₀₀ of 0.7 was obtained. IPTG was then added to a finalconcentration of 0.1 mM for induction. The cells were grown for anadditional 4 hours at a lower temperature of 30° C. and harvested bycentrifugation.

Purification of fusion protein: Recombinant dendroaspins were purifiedas follows by suspending the cell pellets in phosphate buffered saline(PBS) buffer (pH 7.4) containing 1% Triton X-100 and the proteaseinhibitors phenylmethylsulfonyl fluoride (1 mM), pepstatin (1 μM),aprotinin (2 μg/ml), trypsin inhibitor from soyabean (1 μg/ml), 1 mMEDTA, and sonicated on ice. The sonicated mixture was centrifuged at7,800× g at 4° C. for 10 min to pellet the cell debris and insolublematerial. Recombinant GST-dendroaspin and GST-modified-dendroaspins fromsupernatants were purified by affinity chromatography onglutathione-Sepharose CL-4B columns by adsorption in PBS containing 150mM NaCl and elution with 50 mM Tris-HCl containing 10 mM reducedglutathione (pH 8.0). Elution of the adsorbed material with glutathione(pH 8.0) resulted in the appearance of a major band migrating at 32 kDa(GST-dendroaspin fusion protein) in polyacrylamide gels. (FIGS. 5A and5B).

The appropriate fractions comprising the 32-kDa fusion proteins werethen digested in the presence of 150 mM NaCl, 1 mM CaCl₂, and Factor Xa(1:100, w/w, Factor Xa:fusion protein) at 4° C. for 24 h. Treatment ofthe purified GST-proteins with Factor Xa released recombinant proteinsmigrating as 7-kDa bands (FIG. 5C), approximating the size ofdendroaspin, and free GST appearing as an intensification of a 28-kDaband on SDS-polyacrylamide gel electrophoresis. The digested mixture wasloaded onto a Vydac C₁₈ reverse-phase HPLC analytical column (TP104) andeluted with a linear gradient of 0-26% acetonitrile (1.78% per min)containing 0.1% trifluoroacetic acid, followed by 26-36% acetonitrile in0.1% trifluoroacetic acid (0.25% per min). When necessary, furtheranalytical columns were run under the same conditions. FIGS. 6A and 6Bshow the elution profile of reverse phase HPLC runs.

Western blotting: Fusion proteins (10 μg) were electrophoresed on anSDS-PAGE (12%) gel and transferred by semi-dry blotting tonitrocellulose membranes using a current density of 0.8 mA/cm². Thenitrocellulose membranes were blocked for 2 h at RT with 5% dried milkin TBS/Tween buffer (20 mM Tris, 500 mM NaCl, 0.1% Tween) followingwhich they were probed with a corresponding primary antibody overnightand then with either anti-rabbit or anti-mouse second antibody linked tohorseradish peroxidase for 2 h. The blots were then developed withaminoethylcarbazole (FIG. 7).

Production of R51 antibody: Peptide 1 (linear amino acid sequence ofPDGF loop) was conjugated to bovine thyroglobulin (Tg) via rabbit serumalbumin (RSA), via the thiol group, using the hetero-bifunctionalcrosslinkers, sulfphosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulpho-SMCC) and γ-maleimidobutyratesuccinimide (GMBS), respectively. The manufacturer's own procedure wasused. The conjugates were made 1 mg/ml in PBS and stored at −20° C.

New Zealand white rabbits were immunised with Tg-peptide 1 (100 μg ofeach per rabbit injected subcutaneously in 1 ml of 1:1 PBS:Freund'sComplete Adjuvant). The rabbits were boosted after 6 and 12 weeks usingthe same dose but in Freund's Incomplete Adjuvant. Final bleeds weretaken 10 days after the last booster injection . The antisera werealiquoted and stored at −20° C.

Production of R38 and R65 antibodies: IgG (R38 and R65) was purifiedfrom the above-mentioned antisera using Protein-G affinitychromatography (Hi-TRAP Protein-G, Pharmacia, Upsala, Sweden) asdescribed by the manufacturers. Purity was checked by SDS-PAGE. The IgGsolutions in PBS were filter sterilised, aliquoted and stored at −20° C.

EXAMPLE 3 Identification of RGD Function (Dendroaspin Activity)

Platelet aggregation: Platelet aggregation was measured by the increasein light transmission as described in Lu et al (1994) Biochem J 304:929-936 and used to identify RGD function. Briefly, platelet rich plasma(PRP) was prepared from citrated human blood from healthy individuals bycentrifugation at 200 g for 15 min. Platelets were prepared from PRP bycentrifugation at 1200 g for 15 min. The pellets were washed andresuspended in adhesion/aggregation buffer (145 mM NaCl, 5 mM Kcl, 1 mMMgCl₂, 2 mM CaCl₂, 10 mM glucose, 3.5 mg/ml BSA and 10 mM HEPES, pH7.35) and adjusted to a count of 3×10⁸/ml. Platelet aggregation (320 μlincubations) was induced with 10 μM ADP in the presence of 1.67 mg/mlfibrinogen and measured using a Payton Dual-Aggregometer linked to achart recorder.

Platelet adhesion: Platelet adhesion was also measured as described tofurther identify RGD function as described in Lu et al (1994) supra.Briefly, 96 well plates were coated overnight at 4° C. with either humanfibrinogen or fibronection reconstituted in PBS (pH 7.4) at appropriateconcentrations (2-10 μg/ml, 100 μl volumes). Platelets were treated withantagonists at appropriate concentrations for 3 min before addition of90 μl of the mixtures to the microtitre plates which were pre-loadedwith 10 μl of 500 μM ADP giving a final concentration of 50 μM and thenumber of adherent platelets was determined by measuring endogenous acidphosphatase using 130 μl/well of developing buffer (sodium acetate, 10mM p-nitrophenyl phosphate, 0.1% Triton X-100, pH 5.5) and read at410/630 nm on an automated plate reader.

Iodination studies of ligands and ligand binding assays: Iodination ofall variants of dendroaspin was performed using EnzymobeadRadioiodination Reagent (Biorad Laboratories) according to themanufacturer's instructions. The binding of ¹²⁵I-labelled disintegrins,dendroaspin and modified dendroaspins to washed platelets was performedunder equilibrium conditions essentially as described previously in Luet al (1994) supra. The incubation mixture contained 300 μl of washedplatelets (3×10⁸/ml), 10 μl agonist (1.75 mM ADP giving a finalconcentration 50 μM), 10 μl ¹²⁵I-labelled protein samples, 5-20 μlresuspension buffer and was made up to a final volume of 350 μl. Inantibody inhibition studies, platelet suspensions were treated withantibody for 30 min prior to exposure to ADP and then the ¹²⁵I-proteinsamples added and the mixture was incubated at RT for a further 60 min.Incubations were terminated by loading the mixture onto a 25% (w/v)sucrose, 1% BSA cushion and centrifugation at 12,000 g for 10 min. Bothplatelet pellets and supernatants were counted to determine the levelsof bound and free ligand. Background binding levels were determined inthe presence of a 50-fold excess of cold protein samples or disintegrinor 10 mM EDTA.

Identification of grafted loops: Identification of the functions of agrafted loop is dependent on the function of the parent proteins of thegrafted loop.

EXAMPLE 4 Assays for Effects of PDGF Loop I in Dendroaspin

Competition ELISAs (CELIA): The relevant quantity of Den-PDGF, or PDGFas competitors were added simultaneously with appropriately dilutedrabbit anti-PDGF anti-serum and incubated for 2 h. Each rabbitanti-serum was used at a dilution within the linear part of the standarddirect ELISA curve and gave an OD of 1.2 to 1.5 at 405 nm, after a 30min incubation with substrate, when assayed against the appropriateRSA-peptide conjugate.

Cell culture and [³H]-thymidine incorporation assay: Human dermalforeskin fibroblasts (used between passage number 7-10) were maintainedin complete DMEM containing 10% FBS. Sub-confluent monolayers from 75cm³ tissue culture flasks were trypsinized, counted and seeded into 24well (Costar) or 48 well (Falcon) flat bottom tissue culture plates atcell densities of 10,000 cells/well, and grown until approximately 65%confluent (2 days). Cells were rendered quiescent by replacing themedium with complete DMEM containing 0.2% or 0.5% FBS for 48-72 h. Allcell cultures were performed at 37° C. in a humidified atmospherecontaining 8% CO₂ in air.

Inhibition of growth factor induced cellular proliferation was broughtabout by replacing the medium, at time zero, with complete DMEMcontaining varying concentrations of the Den-PDGF and peptide P1 (linearsequence of PDGF domain), respectively. The PDGF concentration forinduction was used at a range of 10-20 ng/ml.

[³H]-thymidine (0.3 mCi/c100 μl/well) was added after 20 h-22 h andincubated until hours 27.5-28. Medium was aspirated, cells were washedtwice with cold PBS and fixed by the addition of 500 μl 10% TCA andincubated for 30 min at 4° C. Cells were next washed once with 0.5 ml70% ethanol and stored at −20° C. Cells were then solubilised byaddition of 500 μl of 0.1 N NaOH to each well for 30 min at roomtemperature. Cellular incorporated [³H]-thymidine was quantified byscintillation counting of a 400 μl sample from each well. The percentageof inhibition of cellular proliferation was worked out as:$\frac{\begin{matrix}{( {\lbrack {}^{3}H \rbrack - {{thymidine}\quad {incorporated}\quad {with}\quad {PDGF}}} ) -} \\( {\lbrack {}^{3}H \rbrack - {{thymidine}\quad {incorporated}\quad {with}\quad {PDGF}} + {M^{*}\quad {or}\quad {Pl}}} )\end{matrix}}{\begin{matrix}{( {\lbrack {}^{3}H \rbrack - {{thymidine}\quad {incorporated}\quad {with}\quad {PDGF}}} ) -} \\( {\lbrack {}^{3}H \rbrack - {{thymidine}\quad {incorporated}\quad {with}\quad 0.2\% \quad {FCS}}} )\end{matrix}} \times 100$ M^(*):  denotes  modified  dendroaspins

FIG. 8 shows that the results of tests in which PDGF-dendroaspininhibits PDGF-induced proliferation by 10-34% (there was no inhibitionwhich has been found when using wild-type dendroaspin as a control) inthe range 6.5-60 μM.

EXAMPLE 5 Modified Dendroasiin Containing a Sequence Derived From theFifth EGF-like Domain of Thrombomodulin

Thrombomodulin serves as a receptor of thrombin. Thrombin is atrypsin-like serine protease fulfilling a central role in bothhaemostasis and thrombosis. In the coagulation cascade, thrombin is thefinal key enzyme, proteolytically cleaving fibrinogen to releasefibrinopeptides A and B and generating fibrin monomers which can thenpolymerize to form a haemostatic plug. In addition to fibrinogencleavage, thrombin exerts a positive feedback on its own production bygenerating coagulation factors Va and VIIIa which act as cofactors ofthrombin activation. Factor XIII is also activated by thrombin andcross-links and stabilises the fibrin polymer. Natural anticoagulantmechanisms limit these processes through inhibition by the serpin,antithrombin III and through activation of protein C bythrombin/thrombomodulin complex. Thrombomodulin is a cell receptorlocated on the endothelial cell surface and it binds and alters themolecular specificity of thrombin by decreasing its ability to catalyzeclot formation while converting thrombin into a potent protein Cactivator. Activated protein C destroys factor Va and VIIIa terminatingthe clotting cascade. Thus, the balance between pro- and anti-coagulantmechanisms maintains the normal physiological conditions and allows thelocal generation of thrombin, whilst preventing it from becoming asystemic or potentially dangerous process. Moreover, thrombin alsoactivates platelets and endothelial cells. Upon platelet activation bythrombin, platelets undergo shape change, aggregation and release theirstorage granule contents (eg platelet factor-4, ADP,5-hydroxytryptamine). Thrombin also increases the synthesis andsecretion of thromboxane A₂ and platelet activating factor. Theinteraction of thrombin with endothelial cells also results in thesecretion of various agents (eg tissue plasminogen activator, PDGF,endothelin) as well as the acceleration of protein C activation bindingto thrombomodulin which initiates protein C anticoagulant pathway.

As examples, a sequence derived from the fifth epidermal growth factor(EGF) like domain of thrombomodulin was grafted into dendroaspin. Thethrombin binding affinity of the novel protein was determined on thebasis of inhibition of fibrinogen clotting.

An appropriate plasmid expression vector was made according to theprocedures of Example 1 except that a thrombomodulin domain was insertedinto dendroaspin as shown in FIG. 3A (SEQ ID NO:14) where the sequencegrafted into loop II of dendroaspin is shown.

Expression, isolation and purification of the TM-modified dendroaspinwas also performed in accordance with Example 1 and the RGD function ofthe modified proteins tested as described in Example 2.

Human thrombomodulin has Mr about 100 kDa and consists of an N-terminaldomain that is homologous to the family of C-type Lectins, six tandemlyrepeated epidermal growth factor (EGF)-like domains, a Ser/Thr richdomain, a transmembrane domain, and a short cytoplasmic tail. In aparticular example shown in FIG. 3C (SEQ ID NO:44), we have inserted asequence derived from the fifth EGF-like domain of thrombomodulin intodendroaspin and made additional modifications of appropriate parts ofthe dendroaspin to prevent the steric hindrance which causes loss of itsbiological functions.

As indicated in FIG. 3D the modified dendroaspin molecules have ADP- andthrombin-induced platelet aggregation activities and they also prolongsthe thrombin clotting time.

EXAMPLE 6 Assays for Effects of Thrombomodulin Domain in Dendroaspin

Thrombin-induced fibrinogen clotting time: Thrombin-induced fibrinogenclotting time was determined using an Amelung KC-10 instrument asfollows. 50 μl of thrombin (3.3 μg/ml) in 50 mM Tris-HCL, pH7.5,containing 150 mM NaCl and 5 mM CaCl₂ was mixed with 10 μl of variousconcentrations of both modified (Den-TM) and wild type dendroaspin (as acontrol for comparison). After a 2 min incubation at 37° C., 100 μl offibrinogen (160 μg/ml) in the same buffer was added to determine theclotting time.

Protein C activation: To check whether the Den-TM has any influence onprotein C activation a two stage assay was used as described by Tsianget al (1990) Biochemistry 29: 10602-10612. In the first stage, thrombinand protein C were added to suitable final concentrations in a reactionvolume of 110 μl, with or without wild-type or mutant dendroaspins witheither 2 mM CaCl₂ or 1 mM Na₂ EDTA. Reaction mixtures were incubated for30 min and stopped by addition of antithrombin III and heparin. Theactive protein C generated was assayed by hydrolysis of substrateS-2366.

Thrombin-induced platelet aggregation: Thrombin-induced plateletaggregation in washed platelets for both wild-type and mutantdendroaspins were determined as described in Example 3 above, exceptthrombin was used as an agonist.

Thrombomodulin binding: Thrombomodulin binding was performed asdescribed by Tsiang et al (1990) supra.

EXAMPLE 7 Modified Dendroaspin Containing a Sequence Derived fromGlycoprotein (GP) IB

Glycoprotein (GP) IBα is required for expression of the high affinityα-thrombin binding site on platelets (Marco et al (1994) J Biol Chem269: 6478-6484). This function may be of crucial importance in theinitiation of haemostasis and thrombosis and may play a role in thedevelopment of pathological vascular occlusion. A modified dendroaspinwas engineered which contains a sequence derived from GP IB to createboth thrombin and integrin antagonist activities.

An appropriate plasmid expression vector was made as described inExample 1, except that a glycoprotein IBα domain was inserted into loopI of the dendroaspin molecule as shown in FIGS. 3A (SEQ ID NOS:11, 15 &16) and 3C (SEQ ID NO:45) where the amino acid residues of the grafteddomain are aligned with the full amino acid sequence of dendroaspin.

As summarized in FIG. 3D, the modified dendroaspin molecules inhibitthrombin activity and platelet aggregation.

Assembly and cloning of the gene of dendroaspin containing glyocoproteinIB (GP IB) domain: The gene of dendroaspin containing GP IB domain wasassembled from the fragments (81 mer, 82 mer, 42 mer and 44 mer) ofwild-type gene, after digestion with Bam HI, EcoR I, Hinf I and Hpa II,and a pair of phosphorylated mutagenesis oligos (83 mer and 82 mer). Theexperimental procedures of annealing, ligation, PCR and assembling weresimilar as described previously for Den-PDGF.

Expression, isolation and purification of the GP IBα-modifieddendroaspin was performed as described in Example I and the RGD functionof the molecule assayed in accordance with Example 3.

EXAMPLE 8 Assays for Effects of the GP IB Domain in Dendroaspin

Measurement of thrombin binding to platelets: Thrombin binding to washedplatelets was measured in a calcium-free aggregation/adhesion buffer(see measurement of platelet aggregation in example 3). Washed plateletswere equilibrated at 37° C. for 10 min before the assay and thenincubated with ¹²⁵I-α-thrombin for 10 min at 25° C. Binding as afunction of ligand concentration was determined with a constantconcentration (0.1 nM) of ¹²⁵I-α-thrombin mixed with increasingconcentrations (between 1 and 200 nM) of unlabeled α-thrombin. Thebinding was initiated by the addition of platelets to the thrombinmixture. Platelets and bound thrombin were separated from unboundthrombin after 10 min incubation by centrifugation through a layer of20% sucrose at 12000 g for 4 min (Lu et al (1994) supra).

The effect of dendroaspin containing GP IB domain on thrombin binding toplatelets was evaluated by mixing varying concentrations of the mutantswith the washed platelets and adding a constant concentration of¹²⁵I-α-thrombin. The results were analyzed as described previously (Luet al (1996) supra).

Measurement of platelet aggregation and secretion: The release of ATPfrom the dense granules of platelets was measured by theluciferin-luciferase assay. Washed platelets were resuspended in 0.4 mlcalcium-free aggregation/adhesion buffer at a count of 2.5×10⁸/ml,aliquots of 0.4 ml were measured using a lumiaggregometer (Chrono-LogCorp). 50 μl of luciferin-luciferase reagent was then added, followed bya-thrombin at the final concentration of 0.26 nM; the ensuing release ofATP was determined by recording the change in luminescence, using alumiaggregometer (Chrono-Log Corp). Platelet aggregation was measured asdescribed in Example 3 above. To test the inhibitory effect of mutantdendroaspin containing GP IB domain on ATP release and plateletaggregation, mutant proteins were added and mixed with platelets for 5min at 37° C. before addition of luciferin/luciferase and thrombin.

Amidolytic activity of thrombin and clotting of fibrinogen: Amidolyticactivity of thrombin and clotting of fibrinogen was determined asdescribed in Example 10 below.

EXAMPLE 9 Modified Dendroaslin Containing a Domain of Hirudin

Hirudin, a potent thrombin inhibitor from the bloodsucking leech Hirudomedicinalis, is a single polypeptide chain protein containing 65 aminoacid residues (Maraganore et al (1989) J Biol Chem 264: 8692-8698). Weproduced a modified dendroaspin which includes amino acid residuesAsn⁵²-Leu⁶⁴, or Phe⁴⁵-Gln⁶⁵ from hirudin. The new construct containsboth anti-thrombin binding and platelet anti-adhesive domains.

The plasmid expression vector was made as described in Example 7, exceptthat further mutation site has been made for a minor changecorresponding to the amino acid residues PRP in loop II of dendroaspin.For the minor changes, the Transformer™ Site-Directed mutagenesis kitfrom Clonetech Laboratories was used. The experimental procedures wereperformed according to manufacturer's instructions except that thehirudin domain was grafted into loops I and II of dendroaspin. Themodified molecules are shown in FIGS. 3A (SEQ ID NOS:12-13, 17-19) and3C (SEQ ID NO:43) where the hirudin-derived amino acid residues arealigned with the native dendroaspin sequence. FIG. 3D indicates how themolecule of FIG. 3C (SEQ ID NO:43) delays thrombin clotting time andinhibits ADP or thrombin induced platelet aggregation.

Expression, isolation and purification of the Den-HR was essentially asdescribed in Example 1. The RGD function of the molecule was establishedaccording to the assay methods of Example 3.

EXAMPLE 10 Assays for Effects of Hirudin Domain in Dendroasdin

Amidolytic activity of thrombin and clotting of fibrinogen: Theamidolytic activity of α-thrombin was performed using the chromogenicsubstrate S-2238 (Chromogenix) and α-thrombin at a final concentrationof 0.06 unit/ml. Thrombin releases p-nitroaniline from the substrate andthe rate of this reaction was monitored in microtiter plates at 405 nmusing an automated spectrophotometer (Autoreader III, Ortho DiagnosticSystems). The inhibitory effect of Den-HR on thrombin cleavage of thechromogenic substrate was measured at final concentration between nM-mM.Thrombin and Den-HR were premixed and reactions were initiated by theaddition of substrate. To evaluate the fibrinogen clotting activity 200μl of α-thrombin (final concentration, 1 nM) in 0.05 M Na phosphate, pH6.5 (BSA, final concentration, 1%) was incubated for 5 min at RT andthen 200 μl of normal plasma was added containing 0.011 M trisodiumcitrate as anticoagulant. The time to observe the start of fibrinogenclotting (thrombin time) was measured using an automated coagulometer at37° C. The effect of mutants on fibrinogen clotting by α-thrombin wasdetermined by substituting the HEPES buffer in the first mixture withmutants at a range of concentrations.

EXAMPLE 11 Modified Dendroaspin Containing a Thrombin-based Pentidewhich Blocks the Procoagulant Activity of Thrombin

The binding site for thrombomodulin within human thrombin has beenlocalized to a region in the B-chain of thrombin. Its sequence wasintroduced into dendroaspin as shown in FIG. 3C (SEQ ID NO:46) togenerate a bifunctional molecule which blocks thrombin procoagulantactivities and also inhibit ADP/thrombin-induced platelet aggregation.Functional characterization of this mutant includes the measurement ofthrombin-induced fibrinogen clotting time as described in Example 6 andthe measurement of platelet aggregation induced by ADP/thrombin asdescribed in Examples 3 and 6 above. FIG. 3D summarizes the propertiesof the bifunctional molecule as being able to delay thrombin clottingand inhibits platelet aggregation induced either by ADP or thrombin.

In the case of the molecules of FIG. 3C (SEQ ID NOS:43-46, 7),modification of the loops has become necessary due to a steric effectcaused by the introduction of “foreign” sequence, e.g. when a sequencederived from PDGF introduced into dendroaspin, anti-PDGF activity wasgenerated, but the anti-platelet activity was lost. This steric effectwas designed out by the introduction of a similar RGD-loop as the loopIII of dendroaspin into loop I. The sequences of engineered moleculesbased on dendroaspin scaffold and their functions are summarized infollowing Table 1 and FIG. 1 respectively.

EXAMPLE 12 Site Directed Mutaaenesis of Modified Dendroaspins

Each of the modified dendroaspins described above can be modified aroundthe RGD loop or any other portion of the molecule as is desired by wayof site directed mutagenesis. Procedures used are as described in Lu etal (1996) supra. Possible RGD flanking region modifications are shown inFIG. 3B (SEQ ID NOS:23-42) together with the results of dendroaspinactivity assays. Some modifications increase activity whereas othermodifications negate activity.

EXAMPLE 13 Antithrombotic Activity of Modified Dendroaspins in a GuineaPig Arterial Thrombosis Model

The four modified dendroaspins produced in the above Examples can betested for antithrombotic activity in vivo in a guinea pig model forarterial thrombosis as described in detail by Carteaux J P et al (1995)Circulation 91: 1568-1574. A range of doses of each modified dendroaspinin the range 0.1 mg/kg of body weight to 1 mg/kg can be administeredseparately to animals by intravenous infusion. Control tests of 50-150IU/kg heparin and placebo can be carried out in parallel.

EXAMPLE 14 Activity of Modified Dendroaspins on Cellular ProliferationFollowing Arterial Injury in a Rabbit Athersclerosis Model

Den-PDGF, Den-TM, Den-GP and Den-HR can be tested in the rabbit in an invivo model system as described by Ragosla M et al (1996) Circulation 93:1194-1200. After induction of athersclerosis, rabbits can be infusedintravenously with a dosage of 0.1 mg-0.5 mg/kg of these modifieddendroaspins. A first set of control animals were treatedintra-arterially with a single bolus of heparin (150IU/kg). A secondcontrol set of animals can be set up to receive saline instead ofmodified dendroaspins. Balloon angioplasty is then performed on test andcontrol animals and followed by quantitative angiography, measurement ofactivated partial thrombosplastin time (aPTT), ³H-thymidineincorporation into the injured artery and follow up studies of luminalnarrowing.

EXAMPLE 15 Thrombolytic Activity of Modified Dendroaspins Tested in anin vivo Pig Model System

The model system of Mruk J S et al (1995) Circulation 93: 792-799 can beemployed. Pigs with occlusive thrombi may be administered intravenouslywith 0.1 mg-1 mg/kg of the modified dendroaspins mentioned in Example 12above. A dose of heparin or hirudin (bolus of 100 IU/kg followedinfusion of 20 IU/kg) can be administered to control animals. Animalsreceiving only saline serve as controls.

48 1 67 PRT Elapidae 1 Glu Gly Ile His Ile Glu Gly Arg Arg Ile Cys TyrAsn His Leu Gly 1 5 10 15 Thr Lys Pro Pro Thr Thr Glu Thr Cys Gln GluAsp Ser Cys Tyr Lys 20 25 30 Asn Ile Trp Thr Phe Asp Asn Ile Ile Arg ArgGly Cys Gly Cys Phe 35 40 45 Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr CysCys Glu Ser Asp Lys 50 55 60 Cys Asn Leu 65 2 217 DNA Elapidae CDS(2)..(199) 2 t ggg atc cat atc gaa ggt cgt cgt atc tgc tac aac cat cttggt act 49 Gly Ile His Ile Glu Gly Arg Arg Ile Cys Tyr Asn His Leu GlyThr 1 5 10 15 aaa ccg ccg act act gaa act tgc cag gaa gac tct tgc tacaaa aac 97 Lys Pro Pro Thr Thr Glu Thr Cys Gln Glu Asp Ser Cys Tyr LysAsn 20 25 30 atc tgg act ttc gac aac atc atc cgt cgt ggt tgc ggt tgc ttcact 145 Ile Trp Thr Phe Asp Asn Ile Ile Arg Arg Gly Cys Gly Cys Phe Thr35 40 45 ccg cgt ggt gac atg ccg ggt ccg tac tgc tgc gaa tct gac aaa tgc193 Pro Arg Gly Asp Met Pro Gly Pro Tyr Cys Cys Glu Ser Asp Lys Cys 5055 60 aac ctt tgagaattct cgtgatga 217 Asn Leu 65 3 66 PRT Elapidae 3 GlyIle His Ile Glu Gly Arg Arg Ile Cys Tyr Asn His Leu Gly Thr 1 5 10 15Lys Pro Pro Thr Thr Glu Thr Cys Gln Glu Asp Ser Cys Tyr Lys Asn 20 25 30Ile Trp Thr Phe Asp Asn Ile Ile Arg Arg Gly Cys Gly Cys Phe Thr 35 40 45Pro Arg Gly Asp Met Pro Gly Pro Tyr Cys Cys Glu Ser Asp Lys Cys 50 55 60Asn Leu 65 4 59 PRT Artificial Sequence Description of ArtificialSequence modified dendroaspin 4 Arg Ile Cys Tyr Asn His Leu Gly Thr LysPro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys AsnIle Trp Thr Phe Asp Asn Ile 20 25 30 Ile Arg Arg Gly Cys Gly Cys Phe ThrPro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys CysAsn Leu 50 55 5 60 PRT Artificial Sequence Description of ArtificialSequence modified dendroaspin 5 Arg Ile Cys Phe Thr Pro Arg Gly Asp MetPro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr LysAsn Ile Trp Thr Phe Asp Asn 20 25 30 Ile Ile Arg Arg Gly Cys Gly Cys PheThr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu Ser Asp LysCys Asn Leu 50 55 60 6 59 PRT Artificial Sequence Description ofArtificial Sequence modified dendroaspin 6 Arg Ile Cys Tyr Asn His LeuGly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser CysIle Ser Arg Arg Leu Ile Asp Arg Thr Asn 20 25 30 Ala Asn Phe Leu Cys GlyCys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Cys Glu SerAsp Lys Cys Asn Leu 50 55 7 60 PRT Artificial Sequence Description ofArtificial Sequence modified dendroaspin 7 Arg Ile Cys Phe Thr Pro ArgGly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp SerCys Ile Ser Arg Arg Leu Ile Asp Arg Thr 20 25 30 Asn Ala Asn Phe Leu CysGly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys GluSer Asp Lys Cys Asn Leu 50 55 60 8 62 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 8 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn 20 25 30 Ala AsnPhe Leu Pro Gly Pro Cys Gly Cys Phe Thr Pro Arg Gly Asp 35 40 45 Met ProGly Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 60 9 60 PRTArtificial Sequence Description of Artificial Sequence modifieddendroaspin 9 Arg Ile Cys Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn AlaAsn Phe 1 5 10 15 Leu Cys Gln Glu Asp Ser Cys Tyr Lys Asn Ile Trp ThrPhe Asp Asn 20 25 30 Ile Ile Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg GlyAsp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 5055 60 10 60 PRT Artificial Sequence Description of Artificial Sequencemodified dendroaspin 10 Arg Ile Cys Ile Ser Arg Arg Leu Ile Asp Arg ThrAsn Ala Asn Phe 1 5 10 15 Leu Cys Gln Glu Asp Ser Cys Arg Lys Ile GluIle Val Arg Lys Lys 20 25 30 Ile Ile Arg Arg Gly Cys Gly Cys Phe Thr ProArg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu Ser Asp Lys Cys AsnLeu 50 55 60 11 61 PRT Artificial Sequence Description of ArtificialSequence modified dendroaspin 11 Arg Ile Cys Gly Asp Thr Asp Leu Tyr AspTyr Tyr Pro Glu Glu Asp 1 5 10 15 Thr Glu Cys Gln Glu Asp Ser Cys TyrLys Asn Ile Trp Thr Phe Asp 20 25 30 Asn Ile Ile Arg Arg Gly Cys Gly CysPhe Thr Pro Arg Gly Asp Met 35 40 45 Pro Gly Pro Tyr Cys Cys Glu Ser AspLys Cys Asn Leu 50 55 60 12 59 PRT Artificial Sequence Description ofArtificial Sequence modified dendroaspin 12 Arg Ile Cys Gly Asp Gly AspPhe Glu Glu Ile Pro Glu Glu Tyr Leu 1 5 10 15 Cys Gln Glu Asp Ser CysTyr Lys Asn Ile Trp Thr Phe Pro Arg Pro 20 25 30 Ile Arg Arg Gly Cys GlyCys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Cys Glu SerAsp Lys Cys Asn Leu 50 55 13 60 PRT Artificial Sequence Description ofArtificial Sequence modified dendroaspin 13 Arg Ile Cys Tyr Asn His LeuGly Thr Lys Pro Pro Arg Pro Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser CysGly Asp Gly Asp Phe Glu Glu Ile Pro Glu 20 25 30 Glu Tyr Pro Arg Pro CysGly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys GluSer Asp Lys Cys Asn Leu 50 55 60 14 61 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 14 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Pro Glu Gly Arg Ile Leu Asp Asp Gly Phe 20 25 30 Ile ThrAsp Ile Asp Glu Cys Gly Cys Phe Thr Pro Arg Gly Asp Met 35 40 45 Pro GlyPro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 60 15 60 PRTArtificial Sequence Description of Artificial Sequence modifieddendroaspin 15 Arg Ile Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr ThrGlu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Gly Asp Thr Asp Leu Tyr AspTyr Tyr Pro 20 25 30 Glu Glu Asp Thr Glu Cys Gly Cys Phe Thr Pro Arg GlyAsp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 5055 60 16 63 PRT Artificial Sequence Description of Artificial Sequencemodified dendroaspin 16 Arg Ile Cys Tyr Asn His Leu Gly Thr Lys Pro ProThr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Gly Asp Thr Asp LeuTyr Asp Tyr Tyr Pro 20 25 30 Glu Glu Asp Thr Glu Pro Gly Pro Cys Gly CysPhe Thr Pro Arg Gly 35 40 45 Asp Met Pro Gly Pro Tyr Cys Cys Glu Ser AspLys Cys Asn Leu 50 55 60 17 73 PRT Artificial Sequence Description ofArtificial Sequence modified dendroaspin 17 Arg Ile Cys Phe Thr Pro ArgGly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp SerCys Tyr Lys Asn Ile Trp Thr Phe Asp Asn 20 25 30 Ile Ile Arg Arg Gly CysGly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Phe ProArg Pro Gln Ser His Asn Asp Gly Asp Phe 50 55 60 Glu Glu Ile Pro Glu GluTyr Leu Gln 65 70 18 69 PRT Artificial Sequence Description ofArtificial Sequence modified dendroaspin 18 Arg Ile Cys Tyr Asn His LeuGly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser CysPhe Thr Pro Arg Gly Asp Met Pro Gly Pro 20 25 30 Tyr Cys Gly Cys Phe ThrPro Arg Gly Asp Met Pro Gly Pro Tyr Cys 35 40 45 Phe Pro Arg Pro Gln SerHis Asn Asp Gly Asp Phe Glu Glu Ile Pro 50 55 60 Glu Glu Tyr Leu Gln 6519 72 PRT Artificial Sequence Description of Artificial Sequencemodified dendroaspin 19 Arg Ile Cys Tyr Asn His Leu Gly Thr Lys Pro ProThr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn Ile TrpThr Phe Asp Asn Ile 20 25 30 Ile Arg Arg Gly Cys Gly Cys Phe Thr Pro ArgGly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Phe Pro Arg Pro Gln Ser His AsnAsp Gly Asp Phe Glu 50 55 60 Glu Ile Pro Glu Glu Tyr Leu Gln 65 70 20 74PRT Artificial Sequence Description of Artificial Sequence modifieddendroaspin 20 Arg Ile Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro TyrPro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr Lys Asn Ile Trp ThrPhe Asp Asn 20 25 30 Ile Ile Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg GlyAsp Met Pro 35 40 45 Gly Pro Tyr Cys Pro Gly Pro Glu Cys Pro Glu Cys TyrIle Leu Asp 50 55 60 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu 65 70 21 70PRT Artificial Sequence Description of Artificial Sequence modifieddendroaspin 21 Arg Ile Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr ThrGlu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Phe Thr Pro Arg Gly Asp MetPro Gly Pro 20 25 30 Tyr Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro GlyPro Tyr Cys 35 40 45 Pro Gly Pro Glu Cys Pro Glu Cys Tyr Ile Leu Asp AspGly Phe Ile 50 55 60 Cys Thr Asp Ile Asp Glu 65 70 22 73 PRT ArtificialSequence Description of Artificial Sequence modified dendroaspin 22 ArgIle Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15Cys Gln Glu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30Ile Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45Pro Tyr Cys Pro Gly Pro Glu Cys Pro Glu Cys Tyr Ile Leu Asp Asp 50 55 60Gly Phe Ile Cys Thr Asp Ile Asp Glu 65 70 23 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 23 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Arg Ile Pro Arg Gly Asp Met Pro Asp 35 40 45 Asp ArgCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 24 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 24 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Arg Arg Ala Arg Gly Asp Asn Pro Asp 35 40 45 Asp ArgCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 25 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 25 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Asp 35 40 45 Asp ArgCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 26 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 26 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Asn Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 27 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 27 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Ala Arg Gly Asp Asn Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 28 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 28 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Ala Gly 35 40 45 Ala TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 29 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 29 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Ala Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 30 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 30 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Ala TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 31 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 31 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Ala Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 32 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 32 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Phe Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 33 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 33 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Trp Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 34 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 34 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Ser Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 35 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 35 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Asp Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 36 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 36 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp His Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 37 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 37 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro ArgCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 38 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 38 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro ThrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 39 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 39 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Leu Asp 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 40 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 40 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Asp 35 40 45 Asp TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 41 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 41 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Lys Gly Asp Met Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 42 59 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 42 Arg Ile CysTyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys GlnGlu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn Ile 20 25 30 Ile ArgArg Gly Cys Gly Cys Phe Thr Pro Lys Gly Asp Trp Pro Gly 35 40 45 Pro TyrCys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 43 72 PRT Artificial SequenceDescription of Artificial Sequence modified dendroaspin 43 Arg Ile CysPhe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro CysGln Glu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn 20 25 30 Ile IleArg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly ProTyr Phe Pro Arg Pro Gln Ser His Asn Asp Gly Asp Phe Glu 50 55 60 Glu IlePro Glu Glu Tyr Leu Gln 65 70 44 70 PRT Artificial Sequence Descriptionof Artificial Sequence modified dendroaspin 44 Arg Ile Cys Phe Thr ProArg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu AspSer Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn 20 25 30 Ile Ile Arg Arg GlyCys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Glu CysPro Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile 50 55 60 Cys Thr Asp Ile AspGlu 65 70 45 66 PRT Artificial Sequence Description of ArtificialSequence modified dendroaspin 45 Arg Ile Cys Phe Thr Pro Arg Gly Asp MetPro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr LysAsn Ile Trp Thr Phe Asp Asn 20 25 30 Ile Ile Arg Arg Gly Cys Gly Cys PheThr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Gly Asp Thr Asp Leu TyrAsp Tyr Tyr Pro Glu Glu Asp 50 55 60 Thr Glu 65 46 52 PRT ArtificialSequence Description of Artificial Sequence modified dendroaspin 46 ArgIle Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15Pro Cys Gln Glu Asp Ser Cys Tyr Lys Asn Ile Trp Thr Phe Asp Asn 20 25 30Ile Ile Arg Arg Gly Pro Gly Pro Thr Trp Thr Ala Asn Val Gly Lys 35 40 45Gly Gln Pro Ser 50 47 45 DNA Artificial Sequence Description ofArtificial Sequence bases 921-956 of pGEX-3X 47 atcgaaggtc gtgggatccccgggaattca tcgtgactga ctgac 45 48 11 PRT Artificial Sequence Descriptionof Artificial Sequence amino acids encoded by bases 921-956 of pGEX-3X48 Ile Glu Gly Arg Gly Ile Pro Gly Asn Ser Ser 1 5 10

What is claimed is:
 1. A hybrid dendroaspin-based polypeptide comprising(a) an amino acid sequence having platelet-binding activity, said aminoacid sequence comprising the tripeptide sequence Arg-Gly-Asp (RGD), and(b) at least one additional amino acid sequence selected from the groupconsisting of: platelet derived growth factor (PDGF), glycoprotein IBα,hirudin, thrombomodulin, vascular epidermal growth factor, transforminggrowth factor-β1, basic fibroblast growth factor, angiotensin II, factorVIII and von Willebrand factor.
 2. A polypeptide as in claim 1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,and SEQ ID NO:
 10. 3. A hybrid dendroaspin-based polypeptide comprising(a) an amino acid sequence having platelet-binding activity, said aminoacid sequence comprising the tripeptide sequence Arg-Gly-Asp (RGD), and(b) at least one additional amino acid sequence having platelet bindingactivity.
 4. A hybrid dendroaspin-based polypeptide as in claim 1,wherein said at least one additional amino acid sequence hasanticoagulant activity.
 5. A hybrid dendroaspin-based polypeptide as inclaim 1 or claim 3, wherein said at least one additional amino acidsequence inhibits clotting.
 6. A hybrid polypeptide comprising (a) adendroaspin scaffold having integrin-binding activity and (b) at leastone non-wild-type dendroaspin domain, wherein said at least onenon-wild-type dendroaspin domain is selected from the group consistingof platelet derived growth factor (PDGF), glycoprotein IBα, hirudin,thrombomodulin, vascular epidermal growth factor, transforming growthfactors β1, basic fibroblast growth factor, angiotensin II, factor VIIIand von Willebrand factor.
 7. A hybrid polypeptide comprising (a) adendroaspin scaffold having integritin-binding activity and (b) at leastone non-wild-type dendroaspin domain, said at least one non-wild-typedendroaspin domain conferring platelet binding activity.
 8. A hybridpolypeptide as in claim 7 wherein said at least one non-wild-typedendroaspin domain is divided into two portions separated by at leastone amino acid residue of said dendroaspin scaffold.
 9. A hybridpolypeptide as in claim 7, comprising at least two non-wild-typedendroaspin domains.
 10. A hybrid polypeptide as in claim 9, whereinsaid at least two non-wild-type dendroaspin domains are the same.
 11. Ahybrid polypeptide comprising (a) a dendroaspin scaffold havingintegrin-binding activity and three structural loops comprising loop I,loop II and loop III, said integrin-binding activity being conferred byloop III, and (b) a non-wild-type dendroaspin domain which isincorporated into a region external to said loops and augments thenative dendroaspin amino acid residues in said region and which confersanticoagulant activity.
 12. A hybrid polypeptide as in claim 11 whereinsaid loop III is modified as compared with native dendroaspin byinsertion, deletion or substitution of between one and eight aminoacids.
 13. A hybrid dendroaspin-based polypeptide comprising (a) anamino acid sequence having platelet-binding activity, said amino acidsequence comprising the tripeptide sequence Arg-Gly-Asp (RGD), and (b)at least one additional amino acid sequence having anticoagulantactivity.
 14. A hybrid dendroaspin-based polypeptide comprising (a) anamino acid sequence having platelet-binding activity, said amino acidsequence comprising the tripeptide sequence Arg-Gly-Asp (RGD), and (b)at least one additional amino acid sequence having antithromboticactivity.
 15. A hybrid dendroaspin-based polypeptide comprising (a) anamino acid sequence having platelet-binding activity, said amino acidsequence comprising the tripeptide sequence Arg-Gly-Asp (RGD), and (b)at least one additional amino acid sequence having cell migrationinhibiting activity.
 16. A hybrid dendroaspin-based polypeptidecomprising (a) an amino acid sequence having platelet-binding activity,said amino acid sequence comprising the tripeptide sequence Arg-Gly-Asp(RGD), and (b) at least one additional amino acid sequence havingclotting inhibiting activity.
 17. A hybrid dendroaspin-based polypeptidecomprising (a) an amino acid sequence having platelet-binding activity,said amino acid sequence comprising the tripeptide sequence Arg-Gly-Asp(RGD), and (b) at least one additional amino acid sequence having signaltransduction regulating activity.
 18. A hybrid polypeptide comprising(a) a dendroaspin scaffold having integrin-binding activity and (b) atleast one non-wild-type dendroaspin domain, said at least onenon-wild-type dendroaspin domain conferring anticoagulant activity. 19.A hybrid polypeptide comprising (a) a dendroaspin scaffold havingintegrin-binding activity and (b) at least one non-wild-type dendroaspindomain, said at least one non-wild-type dendroaspin domain conferringcell migration inhibiting activity.
 20. A hybrid polypeptide comprising(a) a dendroaspin scaffold having integrin-binding activity and (b) atleast one non-wild-type dendroaspin domain, said at least onenon-wild-type dendroaspin domain conferring cell proliferationinhibiting activity.
 21. A hybrid polypeptide comprising (a) adendroaspin scaffold having integrin-binding activity and (b) at leastone non-wild-type dendroaspin domain, said at least one non-wild-typedendroaspin domain conferring clotting inhibiting activity.
 22. A hybridpolypeptide comprising (a) a dendroaspin scaffold havingintegrin-binding activity and (b) at least one non-wild-type dendroaspindomain, said at least one non-wild-type dendroaspin domain conferringsignal transduction regulating activity.
 23. A hybrid dendroaspin-basedpolypeptide as in any of claims 53, 13, 14, 15, 16, or 17, wherein saidpolypeptide has three structural loops I, II and III, and wherein saidamino acid sequence having platelet-binding activity comprises thetripeptide sequence Arg-Gly-Asp (RGD) in said loop III.
 24. Apolypeptide as in claim 23, wherein said loop I begins after amino acidresidue 4 of said polypeptide, said loop II begins after amino acidresidue 23 of said polypeptide, and said loop III begins after aminoacid residue 40 of said polypeptide, and wherein said at least oneadditional amino acid sequence is incorporated into a region external tosaid loops.
 25. A polypeptide as in claim 24, wherein the residues insaid region external to said loops are augmented by the additional aminoacid sequence.
 26. A polypeptide as in claim 23, wherein said at leastone additional amino acid sequence is incorporated into (a) said loop Ior said loop II, or (b) both said loop I and said loop II.
 27. Apolypeptide as in claim 23, wherein said at least one additional aminoacid sequence is incorporated into (a) said loop I or said loop III, orboth said loop I and said loop III.
 28. A polypeptide as in claim 23,wherein said at least one additional amino acid sequence is incorporatedinto said loop II.
 29. A polypeptide as in claim 23, wherein said loopIII is modified by insertion, deletion or substitution of between oneand eight amino acid residues.
 30. A hybrid polypeptide as in any ofclaims 7, 18, 19, 20, 21, or 22, wherein said dendroaspin scaffoldcomprises three structural loops comprising loop I, loop II and loopIII.
 31. A hybrid polypeptide as in claim 30, wherein said loop I beginsafter amino acid residue 4 of said polypeptide, said loop II beginsafter amino acid residue 23 of said polypeptide, and said loop IIIbegins after amino acid residue 40 of said polypeptide, and wherein saidat least one non-dendroaspin amino acid sequence is incorporated into aregion external to said loops.
 32. A polypeptide as in claim 31, whereinthe residues in said region external to said loops are augmented by thenon-dendroaspin amino acid sequence.
 33. A hybrid polypeptide as inclaim 30, wherein said at least one non-wild-type dendroaspin domain isincorporated into (a) said loop I or said loop II, or (b) both said loopI and said loop II.
 34. A hybrid polypeptide as in claim 30, whereinsaid at least one non-wild-type dendroaspin domain is incorporated into(a) said loop I or said loop III, or (b) both said loop I and said loopIII.
 35. A hybrid polypeptide as in claim 30, wherein said at least onenon-wild-type dendroaspin domain is incorporated into said loop II. 36.A hybrid polypeptide as in claim 20, wherein said loop III comprises thetripeptide sequence Arg-Gly-Asp (RGD) and wherein said loop III ismodified by insertion, deletion or substitution of one to eight aminoacid residues.
 37. A hybrid polypeptide as in claim 30, wherein said atleast one non-wild-type dendroaspin domain is inserted into a site inthe dendroaspin scaffold selected from the group consisting of betweenamino acid residues 2-16, 21-36, 21-31, 28-32, 9-13, or 21-33.
 38. Ahybrid polypeptide as in claim 30, wherein said at least onenon-wild-type dendroaspin domain is inserted into the dendroaspinscaffold at the end of the dendroaspin scaffold after residue
 50. 39. Amethod of producing a polypeptide comprising culturing a host celltransformed with a plasmid so as to express said polypeptide, extractingsaid polypeptide from a cell culture, and purifying said polypeptidefrom a cell culture extract, wherein said plasmid comprises a nucleicacid encoding the polypeptide as in anyone of claims, 3, 7, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, or
 22. 40. A pharmaceutical compositioncomprising a therapeutically effective amount of a polypeptide of anyone of claims 1, 2-5, 6-10, 11-12, 13-22, 23-29, or 30-38 and apharmaceutically acceptable excipient or carrier.